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Heterologous Overexpression, Purification and Characterisation of an Alcohol Dehydrogenase (ADH2) from Halobacterium sp. NRC-1

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Abstract

Replacement of chemical steps with biocatalytic ones is becoming increasingly more interesting due to the remarkable catalytic properties of enzymes, such as their wide range of substrate specificities and variety of chemo-, stereo- and regioselective reactions. This study presents characterisation of an alcohol dehydrogenase (ADH) from the halophilic archaeum Halobacterium sp. NRC-1 (HsADH2). A hexahistidine-tagged recombinant version of HsADH2 (His-HsADH2) was heterologously overexpressed in Haloferax volcanii. The enzyme was purified in one step by immobilised Ni-affinity chromatography. His-HsADH2 was halophilic and mildly thermophilic with optimal activity for ethanol oxidation at 4 M KCl around 60 °C and pH 10.0. The enzyme was extremely stable, retaining 80 % activity after 30 days. His-HsADH2 showed preference for NADP(H) but interestingly retained 60 % activity towards NADH. The enzyme displayed broad substrate specificity, with maximum activity obtained for 1-propanol. The enzyme also accepted secondary alcohols such as 2-butanol and even 1-phenylethanol. In the reductive reaction, working conditions for His-HsADH2 were optimised for acetaldehyde and found to be 4 M KCl and pH 6.0. His-HsADH2 displayed intrinsic organic solvent tolerance, which is highly relevant for biotechnological applications.

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References

  1. Schmid, A., Dordick, J. S., Hauer, B., Kiener, A., Wubbolts, M., & Witholt, B. (2001). Industrial biocatalysis today and tomorrow. Nature, 409, 258–268.

    Article  CAS  Google Scholar 

  2. Schoemaker, H. E., Mink, D., & Wubbolts, M. G. (2003). Dispelling the myths—biocatalysis in industrial synthesis. Science, 299, 1694–1697.

    Article  CAS  Google Scholar 

  3. Kroutil, W., Mang, H., Edegger, K., & Faber, K. (2004). Recent advances in the biocatalytic reduction of ketones and oxidation of sec-alcohols. Current Opinion in Chemical Biology, 8, 120–126.

    Article  CAS  Google Scholar 

  4. Yakushi, T., & Matsushita, K. (2010). Alcohol dehydrogenase of acetic acid bacteria: Structure, mode of action, and applications in biotechnology. Applied Microbiology and Biotechnology, 86, 1257–1265.

    Article  CAS  Google Scholar 

  5. Gargiulo, S., Arends, I., & Hollmann, F. (2011). A photoenzymatic system for alcohol oxidation. ChemCatChem, 3, 338–342.

    Article  CAS  Google Scholar 

  6. Klibanov, A. M. (2003). Asymmetric enzymatic oxidoreductions in organic solvents. Current Opinion in Biotechnology, 14, 427–431.

    Article  CAS  Google Scholar 

  7. Klibanov, A. M. (2001). Improving enzymes by using them in organic solvents. Nature, 409, 241–246.

    Article  CAS  Google Scholar 

  8. Cainelli, G., Engel, P. C., Galletti, P., Giacomini, D., Gualandi, A., & Paradisi, F. (2005). Engineered phenylalanine dehydrogenase in organic solvents: Homogeneous and biphasic enzymatic reactions. Organic & Biomolecular Chemistry, 3, 4316–4320.

    Article  CAS  Google Scholar 

  9. Danson, M. J., & Hough, D. W. (1997). The structural basis of protein halophilicity. Comparative Biochemistry and Physiology Part A, Physiology, 117, 307–312.

    Article  Google Scholar 

  10. Lanyi, J. K. (1974). Salt-dependent properties of proteins from extremely halophilic bacteria. Bacteriological Reviews, 38, 272–290.

    CAS  Google Scholar 

  11. Timpson, L., Alsafadi, D., Mac Donnchadha, C., Liddell, S., Sharkey, M., & Paradisi, F. (2012). Characterization of alcohol dehydrogenase (ADH12) from Haloarcula marismortui, an extreme halophile from the Dead Sea. Extremophiles, 16, 57–66.

    Article  CAS  Google Scholar 

  12. Timpson, L., Liliensiek, A.-K., Alsafadi, D., Cassidy, J., Sharkey, M., Liddell, S., et al. (2012). A comparison of two novel alcohol dehydrogenase enzymes (ADH1 and ADH2) from the extreme halophile Haloferax volcanii. Applied Microbiology and Biotechnology, 96(6), 1–9.

    Google Scholar 

  13. Alsafadi, D., & Paradisi, F. (2013). Effect of organic solvents on the activity and stability of halophilic alcohol dehydrogenase (ADH2) from Haloferax volcanii. Extremophiles, 44(2), 511–518.

    Google Scholar 

  14. Dyall-Smith, M. (2008). The halohandbook: Protocols for halobacterial genetics (Vol. 7). Boca Raton: CRC Press.

    Google Scholar 

  15. Allers, T., Barak, S., Liddell, S., Wardell, K., & Mevarech, M. (2010). Improved strains and plasmid vectors for conditional overexpression of His-tagged proteins in Haloferax volcanii. Applied and Environmental Microbiology, 76, 1759–1769.

    Article  CAS  Google Scholar 

  16. Wilkinson, G. N. (1961). Statistical estimations in enzyme kinetics. The Biochemical Journal, 80, 324–332.

    CAS  Google Scholar 

  17. Ng, W. V., Kennedy, S. P., Mahairas, G. G., Berquist, B., Pan, M., Shukla, H. D., et al. (2000). Genome sequence of Halobacterium species NRC-1. Proceedings of the National Academy of Sciences of the United States of America, 97, 12176–12181.

    Article  CAS  Google Scholar 

  18. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215, 403–410.

    CAS  Google Scholar 

  19. Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W., et al. (1997). Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Research, 25, 3389–3402.

    Article  CAS  Google Scholar 

  20. Thompson, J. D., Higgins, D. G., & Gibson, T. J. (1994). CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22, 4673–4680.

    Article  CAS  Google Scholar 

  21. Terpe, K. (2003). Overview of tag protein fusions: From molecular and biochemical fundamentals to commercial systems. Applied Microbiology and Biotechnology, 60, 523–533.

    CAS  Google Scholar 

  22. Woodyer, R., van der Donk, W. A., & Zhao, H. (2003). Relaxing the nicotinamide cofactor specificity of phosphite dehydrogenase by rational design. Biochemistry (Mosc)., 42, 11604–11614.

    Article  CAS  Google Scholar 

  23. Oren, A. (1983). A thermophilic amyloglucosidase from. Halobacterium sodomense, a halophilic bacterium from the Dead Sea. Current Microbiology, 8, 225–230.

    Article  CAS  Google Scholar 

  24. Bonete, M. J., Camacho, M. L., & Cadenas, E. (1987). A new glutamate dehydrogenase from Halobacterium halobium with different coenzyme specificity. International Journal of Biochemistry, 19, 1149–1155.

    Article  CAS  Google Scholar 

  25. Bonete, J. M., Camacho, M. L., & Cadenas, E. (1986). Purification and some properties of NAD +-dependent glutamate dehydrogenase from Halobacterium halobium. International Journal of Biochemistry, 18, 785–789.

    Article  CAS  Google Scholar 

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Acknowledgments

FP acknowledges funding under the Science Foundation Ireland Research Framework Programme and the EPA. Kind donations of the strain H. sp. NRC-1 by Prof. P Engel and the expression system H. volcanii (H1209-H1325)/-pTA963 by Dr. T. Allers are gratefully acknowledged.

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Authors and Affiliations

  1. Centre for Synthesis and Chemical Biology, School of Chemistry, University College Dublin, Belfield, Dublin 4, Ireland

    Ann-Kathrin Liliensiek, Jennifer Cassidy, Gabriele Gucciardo, Cliadhna Whitely & Francesca Paradisi

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  1. Ann-Kathrin Liliensiek
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  2. Jennifer Cassidy
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  3. Gabriele Gucciardo
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  4. Cliadhna Whitely
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  5. Francesca Paradisi
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Correspondence to Francesca Paradisi.

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Ann-Kathrin Liliensiek and Jennifer Cassidy contributed equally to this study.

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Liliensiek, AK., Cassidy, J., Gucciardo, G. et al. Heterologous Overexpression, Purification and Characterisation of an Alcohol Dehydrogenase (ADH2) from Halobacterium sp. NRC-1. Mol Biotechnol 55, 143–149 (2013). https://doi.org/10.1007/s12033-013-9666-4

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